In-cell NMR: recent progresses and future challenges DOI Creative Commons
Enrico Luchinat, Lucia Banci

Rendiconti lincei. Scienze fisiche e naturali, Journal Year: 2023, Volume and Issue: 34(3), P. 653 - 661

Published: June 20, 2023

Abstract In-cell NMR, i.e., NMR spectroscopy applied to studying specific macromolecules within living cells, is becoming the technique of choice for structural and mechanistic description proteins nucleic acids increasingly complex cellular environments, as well temporal evolution biological systems over a broad range timescales. Furthermore, in-cell has already shown its potentialities in early steps drug development. In this Perspective, we report some most recent methodological advancements successful applications spectroscopy, focusing particularly on soluble proteins. We show how combination atomic-level characterization with application context can provide crucial insights processes efficacy unprecedented level detail. Finally, discuss main challenges overcome share our vision future developments that will be made possible.

Language: Английский

Multifunctional nanoparticle for cancer therapy DOI Creative Commons
Yan Gao, Kaiyu Wang, Jin Zhang

et al.

MedComm, Journal Year: 2023, Volume and Issue: 4(1)

Published: Jan. 11, 2023

Cancer is a complex disease associated with combination of abnormal physiological process and exhibiting dysfunctions in multiple systems. To provide effective treatment diagnosis for cancer, current strategies simultaneously focus on various tumor targets. Based the rapid development nanotechnology, nanocarriers have been shown to exhibit excellent potential cancer therapy. Compared nanoparticles single functions, multifunctional are believed be more aggressive potent context targeting. However, not simply an upgraded version original function, but involves sophisticated system proper backbone, optimized modification sites, simple preparation method, efficient function integration. Despite this, many well-designed promising therapeutic emerged recently. Here, give detailed understanding analyzation currently developed nanoparticles, their platform structures organic or inorganic backbones were systemically generalized. We emphasized functionalization strategies, which additional functions nanoparticle. also discussed application that involved nanoformulations functional crosstalk. This review thus provides overview construction advances nanoparticles.

Language: Английский

Citations

45
15NRORC: An Azine Labeling Protocol DOI

Zachary A. Tolchin,

Joel M. Smith

Journal of the American Chemical Society, Journal Year: 2024, Volume and Issue: 146(5), P. 2939 - 2943

Published: Jan. 12, 2024

A practical method for the synthesis of 15N-labeled azines with a high degree isotopic enrichment is described. Activation azine heterocycles an electron-deficient arene allows facile substitution nitrogen atom specifically designed reagent that undergoes canonical ANRORC-type mechanism. wide range can be converted to their corresponding 15N isotopologs using this method, and it also dearomative access reduced heterocyclic congeners. short formal 15N-solifenacin accomplished as well demonstrate application generating labeled pharmaceuticals.

Language: Английский

Citations

25
In-cell NMR suggests that DNA i-motif levels are strongly depleted in living human cells DOI Creative Commons
Pavlína Víšková, Eva Ištvánková, Jan Ryneš

et al.

Nature Communications, Journal Year: 2024, Volume and Issue: 15(1)

Published: March 5, 2024

Abstract I-Motifs (iM) are non-canonical DNA structures potentially forming in the accessible, single-stranded, cytosine-rich genomic regions with regulatory roles. Chromatin, protein interactions, and intracellular properties seem to govern iM formation at sites i-motif propensity (iMFPS) human cells, yet their specific contributions remain unclear. Using in-cell NMR oligonucleotide iMFPS models, we monitor iM-associated structural equilibria asynchronous cell cycle-synchronized HeLa cells 37 °C. Our findings show that displaying pH T < 7 under reference vitro conditions occur predominantly unfolded states while those > appear as a mix of folded depending on cycle phase. Comparing these results previous data obtained using an iM-specific antibody (iMab) reveals cycle-dependent has dual origin, concerns only tiny fraction (possibly 1%) propensity. We propose comprehensive model aligning observations from iMab enabling identification capable adopting physiological living cells. suggest many may have biological roles linked states.

Language: Английский

Citations

23
Synthesis of 15N-Pyridines and Higher Mass Isotopologs via Zincke Imine Intermediates DOI
Hillary M. H. Nguyen, D. Thomas, Marie A. Hart

et al.

Journal of the American Chemical Society, Journal Year: 2024, Volume and Issue: 146(5), P. 2944 - 2949

Published: Jan. 16, 2024

Methods to incorporate stable radioisotopes are integral pharmaceutical and agrochemical development. However, despite the prevalence of pyridines in candidate compounds, methods 15N atoms within their structures limited. Here, we present a general approach pyridine 15N-labeling that proceeds via ring-opening NTf-Zincke imines then ring-closure with commercially available 15NH4Cl salts. This process functions on range substituted pyridines, from simple building block-type compounds late-stage labeling complex pharmaceuticals, 15N-incorporation is >95% most cases. The reactivity Zincke imine intermediates also enables deuteration C3- C5-positions, resulting higher mass isotopologs required for LCMS analysis biological fluids during drug

Language: Английский

Citations

21
Cracking the Code: Reprogramming the Genetic Script in Prokaryotes and Eukaryotes to Harness the Power of Noncanonical Amino Acids DOI

Cosimo Jann,

Sabrina Giofrè,

Rajanya Bhattacharjee

et al.

Chemical Reviews, Journal Year: 2024, Volume and Issue: 124(18), P. 10281 - 10362

Published: Aug. 9, 2024

Over 500 natural and synthetic amino acids have been genetically encoded in the last two decades. Incorporating these noncanonical into proteins enables many powerful applications, ranging from basic research to biotechnology, materials science, medicine. However, major challenges remain unleash full potential of genetic code expansion across disciplines. Here, we provide an overview diverse methodologies systems their final applications prokaryotes eukaryotes, represented by

Language: Английский

Citations

16
Direct Expression of Fluorinated Proteins in Human Cells for 19F In-Cell NMR Spectroscopy DOI Creative Commons

Lan B. T. Pham,

Azzurra Costantino, Letizia Barbieri

et al.

Journal of the American Chemical Society, Journal Year: 2023, Volume and Issue: 145(2), P. 1389 - 1399

Published: Jan. 5, 2023

In-cell NMR spectroscopy is a powerful approach to study protein structure and function in the native cellular environment. It provides precious insights into folding, maturation, interactions, ligand binding of important pharmacological targets directly human cells. However, its widespread application hampered by fact that soluble globular proteins often interact with large components, causing severe line broadening conventional heteronuclear experiments. 19F can overcome this issue, as fluorine atoms incorporated be detected simple background-free 1D spectra. Here, we show fluorinated amino acids easily expressed cells employing medium switch strategy. This straightforward allows incorporation different interest, reaching fluorination efficiencies up 60%, confirmed mass spectrometry X-ray crystallography. The versatility shown performing in-cell on several proteins, including those would otherwise invisible 1H-15N NMR. We apply observe interaction between an intracellular target, carbonic anhydrase 2, inhibitors, investigate how formation complex superoxide dismutase 1 chaperone CCS modulates subunit

Language: Английский

Citations

34
Advanced Techniques for Detecting Protein Misfolding and Aggregation in Cellular Environments DOI
Yulong Bai, Shengnan Zhang,

Hui Dong

et al.

Chemical Reviews, Journal Year: 2023, Volume and Issue: 123(21), P. 12254 - 12311

Published: Oct. 24, 2023

Protein misfolding and aggregation, a key contributor to the progression of numerous neurodegenerative diseases, results in functional deficiencies creation harmful intermediates. Detailed visualization this process is paramount importance for improving our understanding disease mechanisms development potential therapeutic strategies. While vitro studies using purified proteins have been instrumental delivering significant insights into protein misfolding, behavior these complex milieu living cells often diverges significantly from such simplified environments. Biomedical imaging performed cell provides cellular-level information with high physiological pathological relevance, surpassing depth attainable through methods. This review highlights variety methodologies used scrutinize within biological systems. includes optical-based methods, strategies leaning on mass spectrometry, in-cell nuclear magnetic resonance, cryo-electron microscopy. Recent advancements techniques notably deepened processes features resulting misfolded species cells. The fields promises catalyze further breakthroughs comprehension interventions.

Language: Английский

Citations

26
Exploring the dynamics and structure of PpiB in living Escherichia coli cells using electron paramagnetic resonance spectroscopy DOI Creative Commons
Yasmin Ben‐Ishay, Yoav Barak,

Akiva Feintuch

et al.

Protein Science, Journal Year: 2024, Volume and Issue: 33(3)

Published: Feb. 15, 2024

The combined effects of the cellular environment on proteins led to definition a fifth level protein structural organization termed quinary structure. To explore implication potential structure for globular proteins, we studied dynamics and conformations Escherichia coli (E. coli) peptidyl-prolyl cis/trans isomerase B (PpiB) in E. cells. PpiB plays major role maturation regulation folded by catalyzing isomerization proline imidic peptide bond. We applied electron paramagnetic resonance (EPR) techniques, utilizing both Gadolinium (Gd(III)) nitroxide spin labels. In addition using standard labeling approaches with genetically engineered cysteines, incorporated an unnatural amino acid achieve Gd(III)-nitroxide orthogonal labeling. probed PpiB's residue-specific X-band continuous wave EPR at ambient temperatures its double electron-electron (DEER) frozen samples. was delivered cells electroporation. report significant decrease induced two chosen positions. These changes could not be reproduced adding crowding agents cell extracts. Concomitantly, broadening distance distribution coli, determined Gd(III)-Gd(III) DEER measurements, as compared solution human HeLa This suggests increase number present cells, possibly due interactions other components, which also contributes reduction mobility presence

Language: Английский

Citations

12
What can molecular assembly learn from catalysed assembly in living organisms? DOI
Zhi‐Chao Lei, Xinchang Wang,

Liulin Yang

et al.

Chemical Society Reviews, Journal Year: 2024, Volume and Issue: 53(4), P. 1892 - 1914

Published: Jan. 1, 2024

We discuss how living organisms utilize the catalysed assembly (catassembly) way to construct and control complex systems with high efficiency selectivity, we can harness catassembly design functional molecular assemblies.

Language: Английский

Citations

9
Progress, Challenges and Opportunities of NMR and XL-MS for Cellular Structural Biology DOI Creative Commons
Zeting Zhang, Qun Zhao, Zhou Gong

et al.

JACS Au, Journal Year: 2024, Volume and Issue: 4(2), P. 369 - 383

Published: Feb. 5, 2024

The validity of protein structures and interactions, whether determined under ideal laboratory conditions or predicted by AI tools such as Alphafold2, to precisely reflect those found in living cells remains be examined. Moreover, understanding the changes interactions response stimuli within cells, both normal disease conditions, is key grasping proteins' functionality cellular processes. Nevertheless, achieving high-resolution identification these presents a technical challenge. In this Perspective, we summarize recent advancements in-cell nuclear magnetic resonance (NMR) vivo cross-linking mass spectrometry (XL-MS) for studying context. Additionally, discuss challenges, opportunities, potential benefits integrating NMR XL-MS future research offer an exhaustive approach proteins their natural habitat.

Language: Английский

Citations

9